1. Development and Characterization of Synthetic Standards: HCV RNA Susan E. Bromley, Ph.D. Director, Product Development Bayer Healthcare Diagnostics 24 May 2005

2. Goals for Synthetic Reference Standards Provide reference standard that: – –has a confirmed identity – –can be reproducibly manufactured – –can be quantitated by an independent, traceable method – –can be independently monitored for stability – –provides a quantitation anchor for the VERSANT® HCV bDNA Assay standardization scheme. Validate use of reference standard: – –reproducible quantitations between reference standard lots and kit lots

3. Purify Cloned DNAConfirm identity by sequencing In vitro transcription reaction Purify Transcript UV Scan Denaturing gel Digest Transcript Sample Hyperchromicity Phosphate Determination Aliquot and store Tracer incorporation Stability monitored Manufacturing StepsQualification Steps

4. Manufacturing and Qualification Cloned plasmid DNA is used as a template for in vitro transcription to produce RNA transcript – –identity of clone confirmed by sequencing In vitro transcription reaction using T7 RNA polymerase – –incorporation of very low-level radioactive nucleoside precursor – –denaturing gel analysis for full-length product

5. Independent RNA Quantitation Three analytical methods – –A 260nm – –Hyperchromatic Shift – –Phosphate Determination Three methods must agree within 20% Value from phosphate determination used – –Purified RNA digested to nucleosides and phosphates with SVPD and CIP – –Phosphate complexed with ammonium molybdate – –A 360nm calibrated to NIST phosphorus anion standard reference material

6. Stability: Northern Blot Analysis of Reference HCV RNA RNA Marker (Kb) 7.46 4.40 2.37 1.35 Full Length Transcripts RNA Markers (Kb) Degraded RNA 123456 7.46 4.40 2.37 1.35

7. Reference Standard transcript of HCV 5’NC and Core gene Quantification of Kit Calibrators Quantification of QC Panels Quantification of WHO Standard Use of HCV Reference Standard at Bayer Diagnostics Quantification Kit Controls Quantification Patient Samples

8. Validations Process validationProcess validation –Three diverse lots manufactured and qualified according to procedures –All specifications exceeded Standardization validationStandardization validation –Three lots of reference standard and three kit lots –Value assignment of kit calibrators –4 replicates of each calibrator in 6 assays, 3 operators and instruments –Variation in value assignment of the kit calibrators due to all factors was less than 5%.

9. Relationship to WHO Standard Perform value assignment experiment as used to assign quantitations to QC panels and kit calibrators Each value assignment experiment for the WHO Standard includes 9 assays with 4 replicates of each of 6 dilutions WHO value assignment was expanded to include 7 different VERSANT® HCV RNA 3.0 (bDNA) kit lots and 4 lots of HCV Reference Standard 5.2 bDNA copies/WHO IU, 8.3%CV

11. Conclusions Synthetic HCV Reference Standard RNA provides stable quantitation anchor for the VERSANT® HCV RNA 3.0 (bDNA) Assay.Synthetic HCV Reference Standard RNA provides stable quantitation anchor for the VERSANT® HCV RNA 3.0 (bDNA) Assay. In use since 2000In use since 2000 Same model for standardization used for HIV and HBV assays.Same model for standardization used for HIV and HBV assays.